The human brain is composed of billions of cells divided into several regions that enable sensory, motor, and cognitive functions. Although each of these regions is comprised of very similar cell types, the differences at the cell type level between these brain regions are not well described, nor is it well understood how these differences emerge. Understanding these differences is crucial in order to accurately to model brain development in organoid systems, and findings of how regional specification occurs within the developing human brain could promote stem cell based therapies for neurological diseases that frequently affect specific brain regions. Two hypotheses exist to explain the regional differences in the brain: the protomap model purports these differences are inherent in the progenitor cells and maturation gradients that generate the cells of the cortex, while the protocortex explanation surmises the brain is uniform in development until connectivity emerges by way of thalamocortical projections. This proposal will test the relative contribution these models by evaluating the regional differences in cortical layer IV, an area with differentiating neurons and connectivity. Aim I of the proposal will evaluate the protomap hypothesis by first generating a single cell RNA- sequencing (scRNA-seq) map of the sensory and association areas of the brain at three different developmental ages. Informatic in silico sorting will be used to identify the cell types that differentiate to layer IV neurons in order to compare cell maturation gradients. Regional differences found from these analyses will provide hypotheses that will be tested experimentally in in vitro culture cerebral organoids. We will seek to make regionally specific layer IV cells in these organoids, using markers from the scRNA-seq to validate the success of this cellular engineering. Aim II will examine the protocortex hypothesis by adding scRNA-seq from the nuclei of the thalamus to the data obtained in Aim I, highlighting the ages immediately before and after thalamocortical innervation. Gene expression differences that emerge regionally during this critical time period will be candidate markers for how connectivity influences cortical arealization. To test if the thalamus is necessary for these gene expression changes, we will establish a novel human co-culture system that has previously been used in mouse, where cortical and thalamic slice cultures are cultured together and the thalamic slices project to the cortical slice. In this system, we can assay if these areal specific gene expression differentiation programs emerge in independent cortical slice culture, or only with co-culturing these systems. We expect that both the protomap and protocortex models explain some portion of the areally distinct differentiation to cortical layer IV cells, and this proposal will enable us to test these hypotheses and validate our findings with in vitro experimental systems.